The general goal of this proposal is to elucidate the molecular mechanisms that govern the formation of otoliths and otoconia, the biominerals in vertebrate inner ears that convey linear accelerations to the sensory hair cells. The proteins of otoliths and otoconia are thought to play a critical role in their formation. However, testing of this hypothesis is hampered by the fact that most of these proteins are not known. The specific aims of this proposal are, therefore, first to determine the sequence of the about two dozen constituent proteins of zebrafish otoliths; second, to examine the proteins' expression patterns; and third, to test whether these proteins are necessary for otolith formation. In humans, otoconial defects are associated with common vestibular deficits, such as benign paroxysmal positional vertigo and possibly Pendred syndrome. The inaccessibility of the human labyrinth, however, calls for an animal model to study the physiology and pathology of otoconia. Zebrafish otoliths are evolutionary orthologs of mammalian otoconia, based on their shared location, function, composition, and general structure, and thus a valid model system. Furthermore, the mechanisms that govern otolith and otoconia formation are likely to be similar, based on the high degree of molecular conservation found between inner-ear structures of zebrafish and mammals. The zebrafish offers numerous experimental advantages for studies of otolith formation, such as simple husbandry, easy observation and manipulation of gene expression, a genomics infrastructure rivaling that of the mouse, and the accessibility of its transparent and externally-developing embryos. The combination of protein purification and sequencing by mass-spectrometry, molecular cloning of cDNAs, in situ hybridization and quantitative RT-PCR, morpholino injections and mutant rescue will lead to an efficient identification of candidate genes and mechanisms for future studies of mammalian otoconia.